Methods and systems for alignment of a subject for medical imaging

ABSTRACT

Methods and systems for alignment of a subject for medical imaging are disclosed, and involve providing a reference image of an anatomical region of the subject, the anatomical region comprising a target tissue, processing the reference image to generate an alignment reference image, displaying the alignment reference image concurrently with real-time video of the anatomical region, and aligning the real-time video with the alignment reference image to overlay the real-time video with the alignment reference image. Following such alignment, the subject may be imaged using, for example, fluorescence imaging, wherein the fluorescence imaging may be performed by an image acquisition assembly aligned in accordance with the alignment.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/248,199 filed Oct. 29, 2015, titled “METHODS AND SYSTEMS FORALIGNMENT OF A SUBJECT FOR MEDICAL IMAGING,” which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This disclosure relates generally to medical imaging, and moreparticularly to facilitating alignment of a subject for medical imaging.

BACKGROUND OF THE INVENTION

Medical imaging relates to techniques and processes of producing avisual representation of an aspect of a subject's body for clinicalanalysis and/or medical intervention. Examples of medical imaginginclude computed tomography, ultrasound, and magnetic resonance imaging(MRI). Medical imaging can also include optical modalities such asfluorescence medical imaging.

In medical imaging applications, careful setup of the subject forimaging is important. Specifically, in instances where image data isgoing to be compared with previous or future images, it is desirable toreproduce the positioning of the imaging means (e.g., the camera orimaging head) in relation to the anatomical region of interest of thesubject, or vice versa, such that the field of view and orientation ofthe region of interest is the same or close to the same relative to theimaging means for all images. Alignment of the positioning in thisfashion helps to ensure reproducibility over time and minimizesinaccuracies in the assessment of subjects undergoing assessments.Furthermore, such alignment techniques facilitate the use ofquantitative image analysis to aid in the comparative assessments.

Currently available technologies for alignment of a subject to be imagedare cumbersome. Typically, positioning of the subject and/or the imagingmeans is based on approximation of the positioning of the subject by theoperator by visual comparison of the subject to a previously acquiredimage. Such an approach may be time consuming; may be difficult toperform when the subject has issues with mobility; and may be affectedby the level of operator experience, visual subjective judgment, and theamount of time the operator takes to perform the setup. Alignmentsachieved using such an approach may be inaccurate or unreliable.Accordingly, less than optimal imaging may result.

SUMMARY OF THE INVENTION

In accordance with some embodiments, there is provided a method foralignment of a subject for medical imaging. The method includesproviding a reference image of an anatomical region of the subject, theanatomical region including a target tissue, processing the referenceimage to generate an alignment reference image, displaying the alignmentreference image concurrently with real-time video of the anatomicalregion, and aligning the real-time video with the alignment referenceimage to overlay the real-time video with the alignment reference image.The method further includes acquiring the real-time video and processingthe real-time video to quantify the target tissue. The quantification ofthe target tissue includes calculating a dimension of the target tissue,which may be applicable to, for example, wound care or to a surgicalintervention. In an embodiment, the reference image comprises a whitelight image. In an embodiment, processing the reference image togenerate the alignment reference image comprises applying transparencyto the reference image.

In accordance with some embodiments, there is provided an alignmentsystem for alignment of a subject for medical imaging. The alignmentsystem includes a camera assembly for image data acquisition, a displayhaving a user interface, a processor configured to communicate with theuser interface, a non-transitory computer-readable storage medium havinginstructions stored thereon to be executed by the processor. Theinstructions cause the processor to perform operations includingproviding a reference image of the anatomical region of the subject, theanatomical region including a target tissue, processing the referenceimage to generate an alignment reference image, displaying on the userinterface the alignment reference image concurrently or simultaneouslywith real-time video of the anatomical region, and aligning thereal-time video with the alignment reference image to overlay thereal-time video with the alignment reference image. The alignment systemmay be used to aid the quantitative image analysis in comparativeassessments over time.

In some embodiments, a method of aligning an image acquisition assemblyis provided, the method comprising: receiving a reference image of ananatomical region of a subject, the anatomical region comprising atarget tissue; processing the reference image to generate an alignmentreference image; displaying the alignment reference image on a displayconcurrently with real-time video of the anatomical region acquired fromthe image acquisition assembly; dynamically updating the displayedreal-time video to reflect adjustments to a current alignment of theimage acquisition assembly relative to the anatomical region of thesubject; and displaying the real-time video and the alignment referenceimage as overlaid with one another when the current alignment of theimage acquisition assembly is aligned with a predefined alignmentassociated with the reference image.

In some embodiments, the method comprises: illuminating, by a lightsource included in the system, the target tissue to induce fluorescenceemission; when the current alignment of the image acquisition assemblyis aligned with a predefined alignment associated with the referenceimage, capturing, by the image acquisition assembly, a time series offluorescence input data from the fluorescence emission, the fluorescenceinput data capturing transit of the fluorescence agent through thetissue, wherein the image acquisition assembly is configured to receivefluorescence images for fluorescence medical imaging.

In some embodiments of the method, the fluorescence medical imagingcomprises quantitative fluorescence imaging, qualitative fluorescenceimaging, or a combination thereof.

In some embodiments of the method, the illuminating and the capturingare initiated in response to determining that the current alignment isaligned with the predefined alignment.

In some embodiments, the method comprises: in response to detecting thatthe current alignment is not aligned with the predefined alignment,ceasing the illuminating and the capturing.

In some embodiments of the method, inducing fluorescence emissioncomprises inducing fluorescence emission from an endogenous fluorophore,from an exogenous fluorescence imaging agent, or a combination thereof.

In some embodiments of the method, the exogenous fluorescence imagingagent comprises indocyanine green (ICG).

In some embodiments, the method comprises: quantifying the fluorescenceinput data.

In some embodiments, the method comprises: while the current alignmentis aligned with the predefined alignment, acquiring the real-time videoand processing the real-time video to quantify the target tissue.

In some embodiments of the method, the acquiring and processing of thereal time video is initiated in response to detecting that the currentalignment is aligned with the predefined alignment.

In some embodiments of the method, quantifying the target tissuecomprises calculating a dimension of the target tissue.

In some embodiments, the method comprises: in response to detecting thatthe current alignment is not aligned with the predefined alignment,ceasing the acquiring and processing of the real-time video.

In some embodiments of the method, acquiring the reference imagecomprises acquiring the reference image from a camera.

In some embodiments of the method, acquiring the reference imagecomprises retrieving stored data representing the reference image.

In some embodiments of the method, the reference image is a white lightimage, a white light-derived image, a fluorescence image, afluorescence-derived image, or a combination of any one or more thereof.

In some embodiments of the method, processing the reference image togenerate the alignment reference image comprises applying transparencyto the reference image.

In some embodiments, the method comprises: displaying an alignmentindicator on the display, wherein the alignment indicator indicates adifference between the current alignment and the predefined alignment,and wherein the alignment indicator is distinct from the real-time videoand the alignment reference image.

In some embodiments of the method, indicating a difference between thecurrent alignment and the predefined alignment comprises displaying aline between a first portion of the alignment reference image depictinga portion of the anatomical region and a corresponding portion of thereal-time video depicting the portion of the anatomical region.

In some embodiments of the method, the line indicates a direction inwhich the current alignment should be adjusted to align with thepredefined alignment.

In some embodiments, the method comprises: in response to detecting thatthe current alignment is aligned with the predefined alignment,displaying on the display a notification that the current alignment isaligned with the predefined alignment, wherein the notification isdistinct from the real-time video and the alignment reference image.

In some embodiments, the method comprises: in response to detecting thatthe current alignment is aligned with the predefined alignment,providing one of an auditory and a haptic notification that the currentalignment is aligned with the predefined alignment.

In some embodiments of the method, the reference image comprises one orboth of a white light image and a processed image.

In some embodiments of the method, the real-time video comprises one orboth of a white light image and a processed image.

In some embodiments, the method comprises: applying an algorithm todetermine that the current alignment is aligned with the predefinedalignment.

In some embodiments of the method, applying the algorithm comprisescalculating an image transformation matrix including a translation and arotation for aligning the current alignment and the predefinedalignment.

In some embodiments, an alignment system is provided, the alignmentsystem comprising: a display; an image acquisition assembly; aprocessor; and memory storing instructions that, when executed by theprocessor, cause the processor to: receive a reference image of ananatomical region of a subject, the anatomical region comprising atarget tissue; process the reference image to generate an alignmentreference image; display the alignment reference image on a displayconcurrently with real-time video of the anatomical region acquired fromthe image acquisition assembly; update the displayed real-time video toreflect adjustments to a current alignment of the image acquisitionassembly relative to the anatomical region of the subject; and displaythe real-time video and the alignment reference image as overlaid withone another when the current alignment of the image acquisition assemblyis aligned with a predefined alignment associated with the referenceimage.

In some embodiments of the system, the instructions cause the processorto: illuminate, by a light source included in the system, the targettissue to induce fluorescence emission; when the current alignment ofthe image acquisition assembly is aligned with a predefined alignmentassociated with the reference image, capture, by the image acquisitionassembly, a time series of fluorescence input data from the fluorescenceemission, the fluorescence input data capturing transit of thefluorescence agent through the tissue, wherein the image acquisitionassembly is configured to receive fluorescence images for fluorescencemedical imaging.

In some embodiments of the system, the fluorescence medical imagingcomprises quantitative fluorescence imaging, qualitative fluorescenceimaging, or a combination thereof.

In some embodiments of the system, the illuminating and the capturingare initiated in response to determining that the current alignment isaligned with the predefined alignment.

In some embodiments of the system, the instructions cause the processorto, in response to detecting that the current alignment is not alignedwith the predefined alignment, cease the illuminating and the capturing.

In some embodiments of the system, inducing fluorescence emissioncomprises inducing fluorescence emission from an endogenous fluorophore,from an exogenous fluorescence imaging agent, or a combination thereof.

In some embodiments of the system, the exogenous fluorescence imagingagent comprises indocyanine green (ICG).

In some embodiments of the system, the instructions cause the processorto quantify the fluorescence input data.

In some embodiments of the system, the instructions cause the processorto, while the current alignment is aligned with the predefinedalignment, acquire the real-time video and process the real-time videoto quantify the target tissue.

In some embodiments of the system, the acquiring and processing of thereal time video is initiated in response to detecting that the currentalignment is aligned with the predefined alignment.

In some embodiments of the system, quantifying the target tissuecomprises calculating a dimension of the target tissue.

In some embodiments of the system, wherein the instructions cause theprocessor to, in response to detecting that the current alignment is notaligned with the predefined alignment, cease the acquiring andprocessing of the real-time video.

In some embodiments of the system, acquiring the reference imagecomprises acquiring the reference image from a camera.

In some embodiments of the system, acquiring the reference imagecomprises retrieving stored data representing the reference image.

In some embodiments of the system, the reference image is a white lightimage, a white light-derived image, a fluorescence image, afluorescence-derived image, or a combination of any one or more thereof.

In some embodiments of the system, processing the reference image togenerate the alignment reference image comprises applying transparencyto the reference image.

In some embodiments of the system, the instructions cause the processorto display an alignment indicator on the display, wherein the alignmentindicator indicates a difference between the current alignment and thepredefined alignment, and wherein the alignment indicator is distinctfrom the real-time video and the alignment reference image.

In some embodiments of the system, indicating a difference between thecurrent alignment and the predefined alignment comprises displaying aline between a first portion of the alignment reference image depictinga portion of the anatomical region and a corresponding portion of thereal-time video depicting the portion of the anatomical region.

In some embodiments of the system, the line indicates a direction inwhich the current alignment should be adjusted to align with thepredefined alignment.

In some embodiments of the system, the instructions cause the processorto, in response to detecting that the current alignment is aligned withthe predefined alignment, display on the display a notification that thecurrent alignment is aligned with the predefined alignment, wherein thenotification is distinct from the real-time video and the alignmentreference image.

In some embodiments of the system, the instructions cause the processorto, in response to detecting that the current alignment is aligned withthe predefined alignment, provide one of an auditory and a hapticnotification that the current alignment is aligned with the predefinedalignment.

In some embodiments of the system, the reference image comprises one orboth of a white light image and a processed image.

In some embodiments of the system, the real-time video comprises one orboth of a white light image and a processed image.

In some embodiments of the system, the instructions cause the processorto apply an algorithm to determine that the current alignment is alignedwith the predefined alignment.

In some embodiments of the system, applying the algorithm comprisescalculating an image transformation matrix including a translation and arotation for aligning the current alignment and the predefinedalignment.

In some embodiments, a non-transitory computer readable storage mediumis provided, the non-transitory computer readable storage medium storinginstructions, wherein the instructions are executable by a system havinga display, an image acquisition assembly, and a processor to cause thesystem to: receive a reference image of an anatomical region of asubject, the anatomical region comprising a target tissue; process thereference image to generate an alignment reference image; display thealignment reference image on a display concurrently with real-time videoof the anatomical region acquired from the image acquisition assembly;update the displayed real-time video to reflect adjustments to a currentalignment of the image acquisition assembly relative to the anatomicalregion of the subject; and display the real-time video and the alignmentreference image as overlaid with one another when the current alignmentof the image acquisition assembly is aligned with a predefined alignmentassociated with the reference image.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to: illuminate, by a lightsource included in the system, the target tissue to induce fluorescenceemission; when the current alignment of the image acquisition assemblyis aligned with a predefined alignment associated with the referenceimage, capture, by the image acquisition assembly, a time series offluorescence input data from the fluorescence emission, the fluorescenceinput data capturing transit of the fluorescence agent through thetissue, wherein the image acquisition assembly is configured to receivefluorescence images for fluorescence medical imaging.

In some embodiments of the non-transitory computer readable storagemedium, the fluorescence medical imaging comprises quantitativefluorescence imaging, qualitative fluorescence imaging, or a combinationthereof.

In some embodiments of the non-transitory computer readable storagemedium, the illuminating and the capturing are initiated in response todetermining that the current alignment is aligned with the predefinedalignment.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to, in response todetecting that the current alignment is not aligned with the predefinedalignment, cease the illuminating and the capturing.

In some embodiments of the non-transitory computer readable storagemedium, inducing fluorescence emission comprises inducing fluorescenceemission from an endogenous fluorophore, from an exogenous fluorescenceimaging agent, or a combination thereof.

In some embodiments of the non-transitory computer readable storagemedium, the exogenous fluorescence imaging agent comprises indocyaninegreen (ICG).

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to quantify thefluorescence input data.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to, while the currentalignment is aligned with the predefined alignment, acquire thereal-time video and process the real-time video to quantify the targettissue.

In some embodiments of the non-transitory computer readable storagemedium, the acquiring and processing of the real time video is initiatedin response to detecting that the current alignment is aligned with thepredefined alignment.

In some embodiments of the non-transitory computer readable storagemedium, quantifying the target tissue comprises calculating a dimensionof the target tissue.

In some embodiments of the non-transitory computer readable storagemedium, wherein the instructions cause the processor to, in response todetecting that the current alignment is not aligned with the predefinedalignment, cease the acquiring and processing of the real-time video.

In some embodiments of the non-transitory computer readable storagemedium, acquiring the reference image comprises acquiring the referenceimage from a camera.

In some embodiments of the non-transitory computer readable storagemedium, acquiring the reference image comprises retrieving stored datarepresenting the reference image.

In some embodiments of the non-transitory computer readable storagemedium, the reference image is a white light image, a whitelight-derived image, a fluorescence image, a fluorescence-derived image,or a combination of any one or more thereof.

In some embodiments of the non-transitory computer readable storagemedium, processing the reference image to generate the alignmentreference image comprises applying transparency to the reference image.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to display an alignmentindicator on the display, wherein the alignment indicator indicates adifference between the current alignment and the predefined alignment,and wherein the alignment indicator is distinct from the real-time videoand the alignment reference image.

In some embodiments of the non-transitory computer readable storagemedium, indicating a difference between the current alignment and thepredefined alignment comprises displaying a line between a first portionof the alignment reference image depicting a portion of the anatomicalregion and a corresponding portion of the real-time video depicting theportion of the anatomical region.

In some embodiments of the non-transitory computer readable storagemedium, the line indicates a direction in which the current alignmentshould be adjusted to align with the predefined alignment.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to, in response todetecting that the current alignment is aligned with the predefinedalignment, display on the display a notification that the currentalignment is aligned with the predefined alignment, wherein thenotification is distinct from the real-time video and the alignmentreference image.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to, in response todetecting that the current alignment is aligned with the predefinedalignment, provide one of an auditory and a haptic notification that thecurrent alignment is aligned with the predefined alignment.

In some embodiments of the non-transitory computer readable storagemedium, the reference image comprises one or both of a white light imageand a processed image.

In some embodiments of the non-transitory computer readable storagemedium, the real-time video comprises one or both of a white light imageand a processed image.

In some embodiments of the non-transitory computer readable storagemedium, the instructions cause the processor to apply an algorithm todetermine that the current alignment is aligned with the predefinedalignment.

In some embodiments of the non-transitory computer readable storagemedium, applying the algorithm comprises calculating an imagetransformation matrix including a translation and a rotation foraligning the current alignment and the predefined alignment.

In some embodiments, a kit for aligning an image acquisition assembly isprovided, the kit comprising any of the systems described herein and afluorescence imaging agent.

In some embodiments, a fluorescence imaging agent is provided, thefluorescence imaging agent for use in any of the methods or in any ofthe systems described herein for aligning an image acquisition assembly.

In some embodiments of the fluorescence imaging agent, aligning theimage acquisition assembly comprising aligning during for blood flowimaging, tissue perfusion imaging, lymphatic imaging, or a combinationthereof.

In some embodiments of the fluorescence imaging agent, blood flowimaging, tissue perfusion imaging, and/or lymphatic imaging comprisesblood flow imaging, tissue perfusion imaging, and/or lymphatic imagingduring an invasive surgical procedure, a minimally invasive surgicalprocedure, or during a non-invasive surgical procedure.

In some embodiments of the fluorescence imaging agent, the invasivesurgical procedure comprises a cardiac-related surgical procedure or areconstructive surgical procedure.

In some embodiments of the fluorescence imaging agent, thecardiac-related surgical procedure comprises a cardiac coronary arterybypass graft (CABG) procedure.

In some embodiments of the fluorescence imaging agent, wherein the CABGprocedure is on pump or off pump.

In some embodiments, any of the methods, systems, computer-readablestorage media, kits, imaging agents, or other embodiments describedherein may be used for aligning an image acquisition assembly forlymphatic imaging.

In some embodiments, any of the methods, systems, computer-readablestorage media, kits, imaging agents, or other embodiments describedherein may be used for aligning an image acquisition assembly for bloodflow imaging, tissue perfusion imaging, or a combination thereof.

It will be appreciated that any of the variations, aspects, features andoptions described in view of the systems apply equally to the methodsand vice versa. It will also be clear that any one or more of the abovevariations, aspects, features and options can be combined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C illustrate a schematic example of a method according tosome embodiments;

FIG. 2 illustrates an exemplary clinical application of the methodaccording to some embodiments;

FIG. 3 illustrates an example alignment reference image according tosome embodiments;

FIGS. 4A to 4E illustrate example alignment of real-time video data withan alignment reference image according to some embodiments;

FIG. 5 illustrates an example fluorescence imaging system for use inacquiring data derived from fluorescence medical imaging according tosome embodiments;

FIG. 6 illustrates an example illumination module of the fluorescenceimaging system according to some embodiments; and

FIG. 7 illustrates an example camera module of the fluorescence imagingsystem according to some embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to implementations and embodimentsof various systems, methods, and techniques, examples of which areillustrated in the accompanying drawings. Various methods of aligning animage, alignment systems, and non-transitory computer readable storagemedia storing instructions are described herein. Although at least twovariations of the methods of aligning an image, alignment systems,non-transitory computer readable storage media, kits are described,other variations may include aspects of the systems, methods, andnon-transitory computer readable storage media described herein combinedin any suitable manner having combinations of all or some of the aspectsdescribed.

The time between acquisition of image data for a subject can be variable(e.g., hours, days, or months). In order to compare image data acquiredover time, areas being imaged may need to be aligned prior to initiatingimage acquisition. For example, the distance, angle, and rotation of theimaging means relative to the area being imaged should, in someembodiments, be substantially the same for all the image data acquired.According to the various aspects, the methods and systems for opticalimaging use a reference image from previously acquired image data as a“ghosted” (e.g., semi-transparent, translucent, or outlined) overlay,over live (real-time video) image data to assist the imaging deviceoperator with achieving reproducible alignment of the anatomical site ofthe subject.

In accordance with some embodiments, there is provided a method foralignment of a subject for medical imaging using optical modalities,such as, for example, fluorescence imaging.

In accordance with some embodiments, a method of aligning an imageacquisition assembly is provided, the method comprising: receiving areference image of an anatomical region of a subject, the anatomicalregion comprising a target tissue; processing the reference image togenerate an alignment reference image; displaying the alignmentreference image on a display concurrently with real-time video of theanatomical region acquired from the image acquisition assembly;dynamically updating the displayed real-time video to reflectadjustments to a current alignment of the image acquisition assemblyrelative to the anatomical region of the subject; and displaying thereal-time video and the alignment reference image as overlaid with oneanother when the current alignment of the image acquisition assembly isaligned with a predefined alignment associated with the reference image.

The methods and systems for aligning the subject according to thevarious embodiments may aid in qualitative and/or quantitative imageanalysis such as, for example, qualitative and/or quantitative imageanalysis in wound care. In this regard, for example, a change in thewound over time, such as a change in wound dimensions (e.g., diameter,area), or a change in tissue perfusion in the wound and/or around theperi-wound, may be more reproducibly, reliably, and/or accuratelytracked over time with the application of the methods and systems foraligning the subject.

The methods and systems for aligning the subject according to thevarious embodiments may also aid in qualitative and/or quantitativeimage analysis of other clinical imaging applications, such asqualitative and/or quantitative image analysis of surgical interventionsand/or assessments. The methods and systems for alignment may be used,for example, to aid in alignment of successive images intra-operativelyin one surgical intervention or assessment, or to aid in alignment ofimages in multiple surgical interventions or assessments, such as aninitial intervention and/or assessment and one or more follow upinterventions and/or assessments. For example, multiple intra-operativeimages may be recorded and qualitatively and/or quantitatively comparedor collectively analyzed, with the aid of the methods and systems foralignment, during cardiac-related surgery (e.g., cardiac coronary arterybypass graft (CABG) surgery on and/or off pump), cardiovascular surgery,gastrointestinal surgery, plastic/reconstructive surgery (e.g., flapprocedures), lymphatic imaging surgery, or any surgery or any forms ofinvasive surgical procedures where comparison and/or analysis ofmultiple intra-operative images at various time points may be useful. Inthis regard, for example, a change in the target tissue over time(either on a short time scale, intra-operatively, or a longer scale,between successive surgical interventions or assessments) such as achange in perfusion in the tissue and/or around the target tissue (e.g.,in the peri-wound), may be more reproducibly, reliably, and accuratelytracked over time with the application of the methods and systems foraligning the subject.

As is schematically illustrated in FIGS. 1A to 1C, in variousembodiments, the reference image of the anatomical region of the subjectcomprising the target tissue to be imaged may be derived from acquiredreference image data of the anatomical region (e.g., reference imagedata sequence, real-time image data). As shown in FIG. 1A, at element102, real-time image data may be acquired. For example, a camera orother image capture device may acquire real-time image data of theanatomical region. As shown by element 104, reference image data may beacquired in accordance with the real-time image data, such as bycapturing and/or acquiring a reference image by a camera, image sensor,or other image-capture device. In some embodiments, reference image datamay be received or retrieved from computer storage or from a thirdparty, rather than or in addition to being captured or acquired inaccordance with real-time image data. In some embodiments, the referenceimage may be, for example, a single frame selected from the referenceimage data. In various other embodiments, the reference image may bederived from a plurality of frames. For example, the reference image maybe a composite or a mosaic of a plurality of reference image frames, ora reference image sequence. In various embodiments, the reference imagedata (e.g., the reference image) may be acquired and/or generated (e.g.,when the reference image data is processed raw data) during an initialassessment of the subject, or during an assessment chosen as a referenceassessment for subsequent imaging. As shown at element 106, thereference image may be saved or stored for retrieval at a later time,such as during future imaging sessions. As shown at elements 108 and110, the reference image (e.g., the saved or stored reference image)further processed to generate the alignment reference image (FIG. 1B).The resulting alignment reference image or a portion thereof may definethe alignment to be used on subsequent image data acquisitions, and maybe user-selectable. The processing of the reference image to generatethe alignment reference image may, for example, comprise applyingvarious levels of transparency to the reference image to generate asemi-transparent or translucent alignment reference image, it maycomprise processing the reference image to generate an image comprisinghighlighted outlines or contours, it may comprise other image processingto generate an alignment reference image suitable to be viewed by a userwho is simultaneously viewing subsequent-image data, or a combinationthereof. In some embodiments, the alignment reference image may be anoutline or contour of the reference image. In some embodiments, thealignment reference image may be a portion of the reference image. Insome embodiments, the alignment reference image may be a shape generatedby 3D sensors. In various embodiments, the alignment reference image maybe further processed to incorporate or remove color or any otherfeatures as to make it more visually distinct compared to the videoimage data, for example by increasing or decreasing saturation, byfalsely colorizing, by removing color and converting an the alignmentreference image to grayscale or black and white, by brightening ordarkening, or by increasing or decreasing the prominence of one or morecolors in the alignment reference image.

In various embodiments, the reference image may be previously stored andretrievable for use in the methods and systems discussed herein. Inother embodiments, the methods and systems may further involve acquiringthe reference image data for providing the reference image and thealignment reference image.

A skilled person will appreciate that although the methods herein aredescribed in the context of processing a reference image to generate analignment reference image by, for example, applying a level oftransparency to the reference image, a real-time video or a portionthereof may be processed in a similar manner instead of processing areference image. Thus in some embodiments, a level of transparency orother image processing techniques may be applied to a real-time video asopposed to a reference image.

In some embodiments, the alignment reference image or a portion thereofmay be displayed to the user as a static or fixed image concurrently orsimultaneously with live (real-time video) data of the anatomical regionof the subject comprising the target tissue to be imaged duringsubsequent imaging. This technique is schematically illustrated inelement 112 of FIG. 1C, and is further illustrated in the clinicalexample in FIG. 2, where the solid white arrow indicates the alignmentreference image of the subject's foot, and the pattern arrow indicatesvideo of image data misaligned (with the dotted double arrow indicatingthe misalignment) with the alignment reference image. The alignmentreference image may be displayed to the user, for example, as abackground image or as an overlay, or as a partially transparent ortranslucent image. With both the alignment reference image and livevideo data displayed, the user may align the video data until itgenerally overlays the alignment reference image, as shown schematicallyin element 114 of FIG. 1C. As shown in element 116, when alignment isachieved, the user may begin acquisition of the imaging data (e.g.,fluorescence imaging data) as is described in more detail in theExamples section. Acquisition as contemplated by element 116 mayinclude, in some embodiments, capturing and/or storing image data beyondmerely transiently capturing the image data for the purpose ofdisplaying real-time video data for alignment. In some embodiments,acquisition as contemplated in element 116 may include capturing whitelight images and/or fluorescence images, in addition to any other imagetypes contemplated herein. According to some embodiments, image dataacquisition may be triggered to begin automatically once a quantifiedand/or desired level of alignment is achieved. For example, a system mayverify that sufficient alignment has been achieved by measuring a linearmisalignment and determining whether the linear misalignment is below apredefined threshold distance, or by dynamically calculating asimilarity score by comparing the live data and the alignment referenceimage, and determining whether the similarity score is above apredefined threshold. When sufficient alignment is achieved, the systemmay alert the user (e.g., by displaying an indication) and mayautomatically begin image data acquisition. In some embodiments, whensufficient alignment is achieved, then it may be said that a currentalignment corresponding to live video is “aligned” with a predefinedalignment corresponding to the alignment reference image. If sufficientalignment is not achieved between the current alignment and thepredefined alignment, then it may be said that the current alignment andpredefined alignment are not “aligned.” In some embodiments, sufficientalignment may be determined in accordance with any of the techniquesdiscussed above in this paragraph, it may require that optimal alignmentaccording to a sensitivity of a system is achieved (e.g., “exact”alignment according to precision of instrumentation), and/or it mayrequire that alignment is achieved within a predefined threshold orpercentage (e.g., approximate alignment).

While FIGS. 1A to 1C schematically illustrate “WL” (e.g., white light)images, a person of ordinary skill in the art would appreciate that thetechniques described with reference to FIGS. 1A to 1C and elsewhereherein may be equally applicable to other kinds of images and otherkinds of imaging, such as fluorescence imaging. For example, in someembodiments, the real-time image data, reference image, alignmentreference image, and real-time video data may comprise white lightimages, while the acquired video or image data during subsequent imagingmay comprise fluorescence images. Other combinations or arrangements ofwhite light images, fluorescence images, other image types, andcombinations thereof may also be used.

As a result of the alignment, according to various embodiments, theimage data acquired during subsequent imaging generally corresponds in,for example, distance, angle rotation, and/or other parameters a usermay select to the initial reference image data, thus facilitatingrelevant comparison over time that generally corresponds to the sameanatomical location over time. In some embodiments where differentcameras are used to acquire the reference image and the real-time video,the selected parameters of the cameras may be matched as closely aspossible. For example, a field of view of the cameras may besubstantially the same. In some embodiments, there may be somedifferences with respect to resolution of the cameras, or with respectto other properties or parameters of the cameras. A skilled person willappreciate that the more differences there are between the parameters ofthe cameras, the less alignment accuracy may be achieved with respect tothe methods discussed herein. The level of alignment accuracy requiredcan vary depending on the clinical application.

In various embodiments, an alignment indicator may be provided to theuser with regard to an acceptable range of overlap between the alignmentreference image and the video image data during the alignment. Forexample, a boundary or a marker such as a bounding rectangle or cursorwith or without a quantified representation of the degree of alignmentmay be generated and displayed to the user relating to one of or boththe alignment reference image or a portion thereof and the video imagedata within which the video image data must be aligned with thealignment reference image. An example of a boundary rectangle is shownby the white rectangle with rounded corners in FIG. 2. In someembodiments, when an acceptable overlap of bounding rectangles isdetected, the application can automatically change the color of theghosted reference image to indicate to the user that the live video isnow sufficiently aligned with the reference image (e.g., within anacceptable degree of alignment), and to signal to the user that furtheradjustment is not necessary. For example, the ghosted reference imagemay be shown in red when the live video is misaligned with the referenceimage, and after a suitable alignment range is achieved, the referenceimage may turn green. In some embodiments, improved accuracy inautomatic alignment detection may be achieved by utilizing medicaltattoos. In some such embodiments, rather than relying on boundingrectangles, the software can narrow the matching search to the specificshape of the tattoo. Once both shapes overlap within a pre-definedtolerance threshold (e.g., alignment range), feedback (e.g., visual,sound, tactile) can be generated for the user to indicate that thealignment has been achieved and that further adjustment is notnecessary. In some embodiments, the image acquisition system may beconfigured to automatically begin image data acquisition once bothshapes (e.g., rectangles, medical tattoos, etc.) overlap within thepre-defined tolerance threshold. Such an alignment threshold-basedautomated image data acquisition feature may be useful, for example,when the clinical application requires very close image alignment orwhen the application makes a stable camera position difficult tomaintain. In various embodiments, an auto-matching algorithm may be usedas an aid for achieving the alignment, wherein the auto-matchingalgorithm may calculate an alignment score indicative of the alignmentfrom 0% to 100%, with 100% representing the best attainable alignment,or may calculate another quantitative metric of alignment. The alignmentscore may be based, for example, on a calculation of the percentage ofarea of an identified shape or bounding rectangle of a region in thereal-time video, which is overlapping the area of a correspondingmatched shape or bounding rectangle in the reference image. Anacceptable alignment area overlap range may include, for example, analignment overlap range within about 100% to about 99%, about 99% toabout 97%, about 97% to about 95%, about 95% to about 93%, or about 93%to about 90%. As another example, an auto-matching algorithm maycomprise an image registration algorithm that compares image featuresand calculates an optimal best fit alignment of the real-time video andthe reference image, and that calculates a corresponding imagetransformation matrix including translations and rotations that would berequired to correctly align the real-time video to the reference imageor vice-versa. Thresholds may be set for maximum acceptable values oftranslation or rotation values as calculated by the image registrationalgorithm, so that the images may be identified as sufficiently alignedor not sufficiently aligned according to the calculated values.Additionally or alternatively, feedback may be provided to the user toindicate the magnitude and/or direction of mis-alignment between thereal-time image and the reference image, according to the magnitudeand/or direction of the translations and/or rotations as predicted to berequired by the image registration algorithm, such that a user mayexecute the required and indicated rotations and/or translations basedon the provided feedback. In some embodiments, a system having anautomatically-controlled physical component may implement the requiredtranslations or rotations, as determined by an auto-matching algorithm,in response to the required translations or rotations being detected. Askilled person will appreciate that the particular implementation detailin regard to the acceptable alignment overlap range, or the thresholdsfor calculated translation and rotation values, may vary fromapplication to application. For example, in the context of an invasivesurgical procedure such as cardiac bypass surgery, a greater alignmentmay be desirable, for example, to compare images before, during andafter the procedure than, for example, in the context of less invasiveimaging (e.g., wound care).

Various types of image data (e.g., reference image data, video imagedata, or a combination thereof) may be acquired, aligned, viewed, and/orgenerated in accordance with the various embodiments. Examples of theimage data with reference to fluorescence imaging include:

-   -   a color or gray scale white light (WL) image of the anatomy of        the subject comprising the target tissue to be imaged; or    -   a fluorescence image of the target tissue; or    -   processed image data mapped to a false color or range of colors,        such processing and color mapping being used to identify or        emphasize a range of fluorescence intensity; or    -   processed image data mapped to a false color or range of colors,        such processing and color mapping being used to identify or        emphasize changes in fluorescence characteristics over time; or    -   desaturated image data; or    -   a combination of any one or more of the above.

The image data may be either colorized, grayscale, or desaturated andmay comprise one or more regions of interest. In the context of thisspecification, a “color image” of the anatomy of the subject refers to awhite-light image or image sequence or video of the anatomy of thesubject.

In accordance with some embodiments, there is provided an alignmentsystem configured for alignment of a subject for medical imaging. Insome embodiments, an alignment system is provided, the alignment systemcomprising: a display; an image acquisition assembly; a processor; andmemory storing instructions that, when executed by the processor, causethe processor to: receive a reference image of an anatomical region of asubject, the anatomical region comprising a target tissue; process thereference image to generate an alignment reference image; display thealignment reference image on a display concurrently with real-time videoof the anatomical region acquired from the image acquisition assembly;update the displayed real-time video to reflect adjustments to a currentalignment of the image acquisition assembly relative to the anatomicalregion of the subject; and display the real-time video and the alignmentreference image as overlaid with one another when the current alignmentof the image acquisition assembly is aligned with a predefined alignmentassociated with the reference image.

FIG. 3 illustrates an example of the user interface displayed on thedisplay of the alignment system for alignment of a subject for medicalimaging according to an embodiment. The camera assembly of the alignmentsystem (not shown) may be moved by a user into position and heldstationary. In some embodiments, the camera assembly may have a cameraarm (a multi-directional arm) for providing stability for imagealignment, acquisition, or a combination thereof. In some embodiments, acamera arm may be used that features one or more motorized or otherwisecontrollable degrees of freedom of motion of the camera, and automatedmotion of the camera within these controllable degrees of freedom may beperformed to assist with optimizing the image alignment. A skilledperson will appreciate that although the camera arm may be helpful inassisting to keep the camera assembly in a stationary position, it isnot necessary. In various embodiments, the camera assembly can be heldby hand or supported by some other means. In various embodiments, thecamera assembly can be a camera of a medical imaging system (e.g., afluorescence medical imaging system) as is discussed in the exemplaryembodiments below, or it can be another camera assembly imaging in thesame optical imaging plane as the camera of the medical imaging system.In some embodiments, the camera assembly may be configured to provide 3Dimaging.

As is illustrated in FIG. 3, an image may be acquired for alignmentpurposes (i.e., a reference image, an alignment reference image orboth). In the example in FIG. 3, the reference image is acquired withNIR illumination (NIR camera assembly), but in various embodiments itcan be taken in any other way that allows for video with substantiallysimilar imaging conditions.

A processor of the alignment system may be configured to executeinstructions stored on local or remote transitory or non-transitorycomputer readable storage medium, and to cause the system to execute anyone or more of the steps described in connection with methods ortechniques herein. In some embodiments, the processor may be configuredto process the reference image to generate an alignment reference imageas was described above. The software may facilitate transforming thereference image into a semi-transparent image (i.e., the alignmentreference image), or transforming the image in any one or more of themanners discussed above. The reference image, the alignment referenceimage, or both may be stored in any suitable storage medium, such aslocal or remote computer memory, hard disk space, RAM, and/or anytransitory or non-transitory computer-readable storage medium, for usein subsequent imaging and/or assessments.

In subsequent imaging (i.e., the next time the patient is being imaged,or during an imaging session thereafter) and/or assessments, thealignment reference image may be retrieved from storage and used foralignment of the video image data. FIGS. 4A to 4E illustrate progressivealignment of video image data (indicated by the pattern arrow) with thealignment reference image (indicated by the solid white arrow) using analignment system in accordance with the description herein, where thealignment in FIGS. 4A to 4E progressively improves until adequate orsufficient alignment is achieved in FIG. 4E.

Once alignment is achieved (e.g., FIG. 4E), the user can initiateacquisition of the desired image data sequence using the medical imagingsystem, or the system can automatically initiate acquisition upondetecting that sufficient alignment has been achieved. According to someembodiments, another alignment reference image may be acquired duringsuch medical imaging for alignment during further imaging.Alternatively, the alignment reference image from the first assessmentmay be used for all subsequent imaging. Thus, for example, in variousembodiments, the user may choose to align to an alignment referenceimage from the most recent image data sequence acquired, or to the firstalignment reference image from the first image data sequence acquired.The selection of the alignment reference image (e.g., first image orlater acquired image) may depend on clinical circumstances. For example,in some embodiments, using the first-acquired alignment reference imagemay be better from a pure alignment perspective, while the last-acquiredalignment reference image might be better from a practical standpoint asthe anatomy of the subject might change over time, for example, due tosurgery (e.g., amputation, implantation or grafting), wound changes,swelling, other conditions, or a combination of such factors. Multiplealignment reference images may be acquired and stored, in accordancewith one or more of the techniques discussed herein, corresponding tomultiple assessment views of the target tissue, as appropriate ordesired, for example to image different views of the target tissue fromdifferent angles or from different working distances.

In accordance with some embodiments, the alignment system may be astand-alone imaging system or a component of a medical imaging system.For example, in fluorescence medical imaging, the alignment system maybe used with a fluorescence imaging system, or it may be a component ofthe fluorescence imaging system. In an embodiment, the fluorescenceimaging system comprises: a light source configured to illuminate atissue of a subject to induce fluorescence emission from a fluorescenceimaging agent in the tissue; an image acquisition assembly configured toacquire a time series of fluorescence input data from the fluorescenceemission, the fluorescence input data capturing transit of thefluorescence agent through the tissue, and a processor assemblyconfigured to process the time series of fluorescence input data. Insome embodiments, the fluorescence medical imaging system (or othermedical imaging systems) may be considered to be a part of any of thesystems described herein.

Further aspects of such an example fluorescence imaging system aredescribed in more detail in the Examples section.

In yet further aspects, there may be provided a kit including analignment system, a medical imaging system (e.g., a fluorescence medicalimaging system) and an imaging agent (e.g., a fluorescence imagingagent, for example, a fluorescence dye such as ICG or methylene blue).In yet further aspects, there is provided a kit including, a medicalimaging system (e.g., a fluorescence medical imaging system) comprisingan alignment system and an imaging agent wherein the medical imagingsystem is configured to perform the method for alignment of a subjectfor medical imaging. In some embodiments, the alignment system and/ormedical imaging system included in the kit may be any of the alignmentsystems described herein, and/or any system configured to perform any ofthe methods performed herein.

One skilled in the art will appreciate that, although the variousexemplary embodiments are illustrated in the Examples section in thecontext of fluorescence image data, the systems and methods may beapplied to other medical imaging applications comprising the use ofoptical imaging modalities.

EXAMPLES

A Fluorescence Medical Imaging System for Acquisition of Image Data

In some embodiments, a system for alignment of a subject for medicalimaging may be used with or as a component of a medical imaging systemsuch as, for example, a fluorescence medical imaging system foracquiring fluorescence medical image data. An example of such afluorescence medical imaging system is the fluorescence imaging system20 schematically illustrated in FIG. 5. In this embodiment, thefluorescence imaging system 20 is configured to acquire a time series offluorescence signal intensity data (e.g., images, video) capturing thetransit of a fluorescence imaging agent through the tissue.

The fluorescence imaging system 20 (FIG. 5) comprises a light source 22to illuminate the tissue of the subject to induce fluorescence emissionfrom a fluorescence imaging agent 24 in the tissue of the subject (e.g.,in blood), an image acquisition assembly 26 configured to acquire thetime series of fluorescence images from the fluorescence emission, and aprocessor assembly 28 configured to utilize the acquired time series offluorescence images (fluorescence signal intensity data) according tothe various embodiments described herein.

In various embodiments, the light source 22 (FIG. 5) comprises, forexample, an illumination module 30 (FIG. 6) comprising a fluorescenceexcitation source configured to generate an excitation light having asuitable intensity and a suitable wavelength for exciting thefluorescence imaging agent 24. The illumination module 30 in FIG. 6comprises a laser diode 32 (e.g., which may comprise, for example, oneor more fiber-coupled diode lasers) configured to provide an excitationlight to excite the fluorescence imaging agent 24 (not shown). Examplesof other sources of the excitation light which may be used in variousembodiments include one or more LEDs, arc lamps, or other illuminanttechnologies of sufficient intensity and appropriate wavelength toexcite the fluorescence imaging agent 24 in the tissue (e.g., in blood).For example, excitation of the fluorescence imaging agent 24 in blood,wherein the fluorescence imaging agent 24 is a fluorescence dye withnear infra-red excitation and emission characteristics, may be performedusing one or more 793 nm, conduction-cooled, single bar, fiber-coupledlaser diode modules from DILAS Diode Laser Co, Germany.

In various embodiments, the light output from the light source 22 inFIG. 5 may be projected through an optical element (e.g., one or moreoptical elements) to shape and guide the output being used to illuminatethe tissue area of interest. The shaping optics may consist of one ormore lenses, light guides, and/or diffractive elements so as to ensure aflat field over substantially the entire field of view of the imageacquisition assembly 26. In particular embodiments, the fluorescenceexcitation source is selected to emit at a wavelength close to theabsorption maximum of the fluorescence imaging agent 24 (e.g., ICG). Forexample, referring to the embodiment of the illumination module 30 inFIG. 6, the output 34 from the laser diode 32 is passed through one ormore focusing lenses 36, and then through a homogenizing light pipe 38such as, for example, light pipes commonly available from NewportCorporation, USA. Finally, the light is passed through an opticaldiffractive element 40 (e.g., one or more optical diffusers) such as,for example, ground glass diffractive elements also available fromNewport Corporation, USA. Power to the laser diode 32 itself is providedby, for example, a high-current laser driver such as those availablefrom Lumina Power Inc. USA. The laser may optionally be operated in apulsed mode during the image acquisition process. In this embodiment, anoptical sensor such as a solid state photodiode 42 is incorporated intothe illumination module 30 and samples the illumination intensityproduced by the illumination module 30 via scattered or diffusereflections from the various optical elements. In various embodiments,additional illumination sources may be used to provide guidance whenaligning and positioning the module over the area of interest.

Referring back to FIG. 5, in various embodiments, the image acquisitionassembly 26 may be a component of, for example, the fluorescence imagingsystem 20 configured to acquire the time series of fluorescence images(e.g., video) from the fluorescence emission from the fluorescenceimaging agent 24. Referring to FIG. 7, there is shown an exemplaryembodiment of an image acquisition assembly 26 comprising a cameramodule 50. As is shown in FIG. 7, the camera module 50 acquires imagesof the fluorescence emission 52 from the fluorescence imaging agent 24in the tissue (e.g., in blood) (not shown) by using a system of imagingoptics (e.g., front element 56 a, rejection filter 56 b, dichroic 60 andrear element 62) to collect and focus the fluorescence emission onto animage sensor assembly 64 comprising at least one 2D solid state imagesensor. A rejection filter 56 b may be, for example, a notch filter usedto reject a band of wavelengths corresponding to the excitation light. Adichroic 60 may be, for example, a dichroic mirror used to selectivelypass one subset of the incoming light wavelength spectrum and redirectremaining wavelengths off of the optical path for rejection or towards aseparate image sensor. The solid state image sensor may be a chargecoupled device (CCD), a CMOS sensor, a CID or similar 2D sensortechnology. The charge that results from the optical signal transducedby the image sensor assembly 64 is converted to an electrical videosignal, which includes both digital and analog video signals, by theappropriate read-out and amplification electronics in the camera module50.

According to some embodiments, excitation wavelength of about 800nm+/−10 nm and emission wavelengths of >820 nm are used along with NIRcompatible optics for ICG fluorescence imaging. A skilled person willappreciate that other excitation and emission wavelengths may be usedfor other imaging agents.

Referring back to FIG. 5, in various embodiments, the processor assembly28 comprises, for example,

-   -   a processor module (not shown) configured to perform various        processing operations, including executing instructions stored        on computer-readable medium, wherein the instructions cause one        or more of the systems described herein to execute the methods        and techniques described herein, and    -   a data storage module (not shown) to record and store the data        from the operations, as well as to store, in some embodiments,        instructions executable by the processor module to implement the        methods and techniques disclosed herein.

In various embodiments, the processor module comprises any computer orcomputing means such as, for example, a tablet, laptop, desktop,networked computer, or dedicated standalone microprocessor. Inputs aretaken, for example, from the image sensor 64 of the camera module 50shown in FIG. 7, from the solid state photodiode in the illuminationmodule 30 in FIG. 6, and from any external control hardware such as afootswitch or remote-control. Output is provided to the laser diodedriver, and optical alignment aids. In various embodiments, theprocessor assembly 28 (FIG. 5) may have a data storage module with thecapability to save the time series of input data (e.g., image data) to atangible non-transitory computer readable medium such as, for example,internal memory (e.g. a hard disk or flash memory), so as to enablerecording and processing of data. In various embodiments, the processormodule may have an internal clock to enable control of the variouselements and ensure correct timing of illumination and sensor shutters.In various other embodiments, the processor module may also provide userinput and graphical display of outputs. The fluorescence imaging systemmay optionally be configured with a video display (not shown) to displaythe images as they are being acquired or played back after recording, orfurther to visualize the data generated at various stages of the methodas was described above.

In operation, and with continuing reference to the exemplary embodimentsin FIGS. 5 to 7, as a result of the alignment of the subject accordingto the various embodiments, the subject is in a position for imagingwhere the anatomical area of interest of the subject is located beneathboth the light source 22 and the image acquisition assembly 26 such thata substantially uniform field of illumination is produced acrosssubstantially the entire area of interest. In various embodiments, priorto the administration of the fluorescence imaging agent 24 to thesubject, an image may be acquired of the area of interest for thepurposes of background deduction. For example, in order to do this, theoperator of the fluorescence imaging system 20 in FIG. 5 may initiatethe acquisition of the time series of fluorescence images (e.g., video)by depressing a remote switch or foot-control, or via a keyboard (notshown) connected to the processor assembly 28. As a result, the lightsource 22 is turned on and the processor assembly 28 begins recordingthe fluorescence image data provided by the image acquisition assembly26. In lieu of the pulsed mode discussed above, it will be understoodthat, in some embodiments, the light source 22 can comprise an emissionsource which is continuously on during the image acquisition sequence.When operating in the pulsed mode of the embodiment, the image sensor 64in the camera module 50 (FIG. 7) is synchronized to collect fluorescenceemission following the laser pulse produced by the diode laser 32 in theillumination module 30 (FIG. 6). In this way, maximum fluorescenceemission intensity is recorded, and signal-to-noise ratio is optimized.In this embodiment, the fluorescence imaging agent 24 is administered tothe subject and delivered to the area of interest via arterial flow.Acquisition of the time series of fluorescence images is initiated, forexample, shortly after administration of the fluorescence imaging agent24, and the time series of fluorescence images from substantially theentire area of interest are acquired throughout the ingress of thefluorescence imaging agent 24. The fluorescence emission from the regionof interest is collected by the collection optics of the camera module50. Residual ambient and reflected excitation light is attenuated bysubsequent optical elements (e.g., optical element 60 in FIG. 7 whichmay be a filter) in the camera module 50 so that the fluorescenceemission can be acquired by the image sensor assembly 64 with minimalinterference by light from other sources.

In various embodiments, the processor is in communication with theimaging system or is a component of the imaging system. The program codeor other computer-readable instructions, according to the variousembodiments, can be written and/or stored in any appropriate programminglanguage and delivered to the processor in various forms, including, forexample, but not limited to information permanently stored onnon-writeable storage media (e.g., read-only memory devices such as ROMsor CD-ROM disks), information alterably stored on writeable storagemedia (e.g., hard drives), information conveyed to the processor viatransitory mediums (e.g., signals), information conveyed to theprocessor through communication media, such as a local area network, apublic network such as the Internet, or any type of media suitable forstoring electronic instruction. In various embodiments, the tangiblenon-transitory computer readable medium comprises all computer-readablemedia. In some embodiments, computer-readable instructions forperforming one or more of the methods or techniques discussed herein maybe stored solely on non-transitory computer readable media.

In some embodiments, the system and method for alignment of the subjectfor medical imaging may be a component of a medical imaging system suchas the fluorescence medical imaging system 20, which acquires themedical image data and is further configured to align the subject forsuch imaging. In embodiments where the alignment system is a componentof the imaging system, such as the fluorescence imaging system describedabove, the light source, the image acquisition assembly and theprocessor of the imaging system may function as the camera assembly andthe processor of the alignment system. A skilled person will appreciatethat imaging systems other than fluorescence imaging systems may beemployed for use with alignment systems such as those described herein,depending on the type of imaging being performed.

Example Imaging Agents for Use in Generating Image Data

According to some embodiments, in fluorescence medical imagingapplications, the imaging agent is a fluorescence imaging agent such as,for example, indocyanine green (ICG) dye. ICG, when administered to thesubject, binds with blood proteins and circulates with the blood in thetissue. The fluorescence imaging agent (e.g., ICG) may be administeredto the subject as a bolus injection (e.g., into a vein or an artery) ina concentration suitable for imaging such that the bolus circulates inthe vasculature and traverses the microvasculature. In other embodimentsin which multiple fluorescence imaging agents are used, such agents maybe administered simultaneously, e.g. in a single bolus, or sequentiallyin separate boluses. In some embodiments, the fluorescence imaging agentmay be administered by a catheter. In certain embodiments, thefluorescence imaging agent may be administered less than an hour inadvance of performing the measurement of signal intensity arising fromthe fluorescence imaging agent. For example, the fluorescence imagingagent may be administered to the subject less than 30 minutes in advanceof the measurement. In yet other embodiments, the fluorescence imagingagent may be administered at least 30 seconds in advance of performingthe measurement. In still other embodiments, the fluorescence imagingagent may be administered contemporaneously with performing themeasurement.

According to some embodiments, the fluorescence imaging agent may beadministered in various concentrations to achieve a desired circulatingconcentration in the blood. For example, in embodiments where thefluorescence imaging agent is ICG, it may be administered at aconcentration of about 2.5 mg/mL to achieve a circulating concentrationof about 5 μM to about 10 μM in blood. In various embodiments, the upperconcentration limit for the administration of the fluorescence imagingagent is the concentration at which the fluorescence imaging agentbecomes clinically toxic in circulating blood, and the lowerconcentration limit is the instrumental limit for acquiring the signalintensity data arising from the fluorescence imaging agent circulatingwith blood to detect the fluorescence imaging agent. In various otherembodiments, the upper concentration limit for the administration of thefluorescence imaging agent is the concentration at which thefluorescence imaging agent becomes self-quenching. For example, thecirculating concentration of ICG may range from about 2 μM to about 10mM. Thus, in one aspect, the method comprises the step of administrationof the imaging agent (e.g., a fluorescence imaging agent) to the subjectand acquisition of the signal intensity data (e.g., video) prior toprocessing the signal intensity data according to the variousembodiments. In another aspect, the method excludes any step ofadministering the imaging agent to the subject.

According to some embodiments, a suitable fluorescence imaging agent foruse in fluorescence imaging applications to generate fluorescence imagedata is an imaging agent which can circulate with the blood (e.g., afluorescence dye which can circulate with, for example, a component ofthe blood such as lipoproteins or serum plasma in the blood) and transitvasculature of the tissue (i.e., large vessels and microvasculature),and from which a signal intensity arises when the imaging agent isexposed to appropriate light energy (e.g., excitation light energy, orabsorption light energy). In various embodiments, the fluorescenceimaging agent comprises a fluorescence dye, an analogue thereof, aderivative thereof, or a combination of these. A fluorescence dyeincludes any non-toxic fluorescence dye. In certain embodiments, thefluorescence dye optimally emits fluorescence in the near-infraredspectrum. In certain embodiments, the fluorescence dye is or comprises atricarbocyanine dye. In certain embodiments, the fluorescence dye is orcomprises indocyanine green (ICG), methylene blue, or a combinationthereof. In other embodiments, the fluorescence dye is or comprisesfluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, fluorescamine, rose Bengal, trypanblue, fluoro-gold, or a combination thereof, excitable using excitationlight wavelengths appropriate to each dye. In some embodiments, ananalogue or a derivative of the fluorescence dye may be used. Forexample, a fluorescence dye analog or a derivative includes afluorescence dye that has been chemically modified, but still retainsits ability to fluoresce when exposed to light energy of an appropriatewavelength.

In various embodiments, the fluorescence imaging agent may be providedas a lyophilized powder, solid, or liquid. In certain embodiments, thefluorescence imaging agent may be provided in a vial (e.g., a sterilevial), which may permit reconstitution to a suitable concentration byadministering a sterile fluid with a sterile syringe. Reconstitution maybe performed using any appropriate carrier or diluent. For example, thefluorescence imaging agent may be reconstituted with an aqueous diluentimmediately before administration. In various embodiments, any diluentor carrier which will maintain the fluorescence imaging agent insolution may be used. As an example, ICG may be reconstituted withwater. In some embodiments, once the fluorescence imaging agent isreconstituted, it may be mixed with additional diluents and carriers. Insome embodiments, the fluorescence imaging agent may be conjugated toanother molecule, such as a protein, a peptide, an amino acid, asynthetic polymer, or a sugar, for example to enhance solubility,stability, imaging properties, or a combination thereof. Additionalbuffering agents may optionally be added including Tris, HCl, NaOH,phosphate buffer, and/or HEPES.

A person of skill in the art will appreciate that, although afluorescence imaging agent was described above in detail, other imagingagents may be used in connection with the systems, methods, andtechniques described herein, depending on the optical imaging modality.

In some embodiments, the fluorescence imaging agent used in combinationwith the methods and systems described herein may be used for blood flowimaging, tissue perfusion imaging, lymphatic imaging, or a combinationthereof, which may performed during an invasive surgical procedure, aminimally invasive surgical procedure, a non-invasive surgicalprocedure, or a combination thereof. Examples of invasive surgicalprocedure which may involve blood flow and tissue perfusion include acardiac-related surgical procedure (e.g., CABG on pump or off pump) or areconstructive surgical procedure. An example of a non-invasive orminimally invasive procedure includes wound (e.g., chronic wound such asfor example pressure ulcers) treatment and/or management. Examples oflymphatic imaging include identification of one or more lymph nodes,lymph node drainage, lymphatic mapping, or a combination thereof. Insome variations such lymphatic imaging may relate to the femalereproductive system (e.g., uterus, cervix, vulva).

Examples of Image Data Alignment According to an Embodiment

FIG. 2 illustrates example results generated according to the variousembodiments described above relating to an application of the methodsand systems to alignment of a subject for fluorescence imaging. Thefluorescence image data was generated using a fluorescence imagingsystem (available from NOVADAQ® Technologies Inc.), and ICG was used asthe fluorescence imaging agent.

FIG. 2 illustrates image data alignment according to an embodiment wherethe alignment reference image (indicated by the solid white arrow) issimultaneously displayed to the user on a display with video image data(indicated by the pattern arrow) during a subsequent assessment of thesubject following the initial assessment during which the alignmentreference image was generated. The initial, or preceding, assessment maybe from a previous patient visit or interaction (e.g., if assessing thecourse of wound and/or tissue healing over multiple visits), or from anearlier point in time during the same visit (e.g., if taking multipleintra-operative image assessments during a surgical procedure). Thedashed line shows the degree of misalignment or offset of the videoimage data with the alignment reference image. Without correcting forsuch misalignment, the fluorescence image data acquired during thesubsequent assessment would not be suitable for an accurate comparison,including a quantitative assessment, with the image data from theinitial assessment, and any clinical conclusions drawn from such datawould therefore not be accurate. A user, seeing such misalignment(and/or a system detecting such misalignment), based on a comparison ofthe video image data and the simultaneously displayed alignmentreference image, may align the video image data to mimic the imageacquisition conditions from the preceding assessment, as was describedin the various embodiments, without having to rely solely on his or herexperience, judgment, memory, and estimation as to the proper alignment.Instead, the user may be provided with dynamic visual feedback,including the ability to view both the video image data and thealignment reference image at once, and the user further may be providedwith user-interface cues provided by the system including indications ofan offset distance, indication of a metric indicating the strength ofthe current alignment, boundaries or markers to aid achieving alignment,and or express indications (e.g., visual, auditory, or hapticnotifications) that sufficient alignment has or has not been achieved.By leveraging these forms of dynamic visual (and/or auditory and/orhaptic) feedback, a system may achieve accurate alignment more reliablyand more efficiently. Following such alignment, fluorescence imaging canbe initiated as was described above in connection with the variousembodiments. Proper alignment for medical imaging is important as itaids qualitative and quantitative monitoring of clinical outcomes overtime in a controlled manner.

Thus, the methods and systems in accordance with the various embodimentsare intuitive and user friendly and may be used by inexperienced and/orexperienced users to minimize inaccuracies in medical imaging andanalysis, such as for example fluorescence imaging for the analysis ofblood flow dynamics, including tissue perfusion analysis. Tissueperfusion relates to the microcirculatory flow of blood per unit tissuevolume in which oxygen and nutrients are provided to and waste isremoved from the capillary bed of the tissue being perfused. Adistinction should be drawn between vascular blood flow and tissue bloodperfusion, namely that tissue perfusion is a phenomenon related to butalso distinct from blood flow in vessels. Quantified blood flow throughblood vessels may be expressed in terms that define flow (i.e.,volume/time), or that define speed (i.e., distance/time). Tissue bloodperfusion defines movement of blood through micro-vasculature, such asarterioles, capillaries, or venules, within a tissue volume. Quantifiedtissue blood perfusion may be expressed in terms of blood flow throughtissue volume, namely, that of blood volume/time/tissue volume (ortissue mass). Perfusion is associated with nutritive blood vessels(e.g., micro-vessels known as capillaries) that comprise the vesselsassociated with exchange of metabolites between blood and tissue, ratherthan larger-diameter non-nutritive vessels. In some embodiments,quantification of a target tissue may include calculating or determininga parameter or an amount related to the target tissue, such as a rate,size volume, time, distance/time, and/or volume/time, and/or an amountof change as it relates to any one or more of the preceding parametersor amounts. However, compared to blood movement through the largerdiameter blood vessels, blood movement through individual capillariescan be highly erratic, principally due to vasomotion, whereinspontaneous oscillation in blood vessel tone manifests as pulsation inerythrocyte movement.

While the present disclosure has been illustrated and described inconnection with various embodiments shown and described in detail, it isnot intended to be limited to the details shown, since variousmodifications and structural changes may be made without departing inany way from the scope of the present disclosure. Various modificationsof form, arrangement of components, steps, details and order ofoperations of the embodiments illustrated, as well as other embodimentsof the disclosure may be made without departing in any way from thescope of the present disclosure, and will be apparent to a person ofskill in the art upon reference to this description. It is thereforecontemplated that the appended claims will cover such modifications andembodiments as they fall within the true scope of the disclosure. Forthe purpose of clarity and a concise description, features are describedherein as part of the same or separate embodiments, however, it will beappreciated that the scope of the disclosure includes embodiments havingcombinations of all or some of the features described. For the terms“for example” and “such as,” and grammatical equivalences thereof, thephrase “and without limitation” is understood to follow unlessexplicitly stated otherwise. As used herein, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise.

The invention claimed is:
 1. A method of aligning a camera, the methodcomprising: receiving a reference image of an anatomical region of asubject, the anatomical region comprising a target tissue; processingthe reference image to generate an alignment reference image; displayingthe alignment reference image on a display concurrently with real-timevideo of the anatomical region acquired from the camera; dynamicallyupdating the displayed real-time video to reflect adjustments to acurrent alignment of the camera relative to the anatomical region of thesubject; and displaying the real-time video and the alignment referenceimage as overlaid with one another when the current alignment of thecamera is aligned with a predefined alignment associated with thereference image; determining that the current alignment is aligned withthe predefined alignment, wherein the determination is performed beforeinitiating illuminating, by a light source, of the target tissue toinduce fluorescence emission and is performed before initiatingcapturing, by the camera, of a time series of fluorescence input datafrom the fluorescence emission; in response to the determining that thecurrent alignment is aligned with the predefined alignment: initiatingthe illuminating, by the light source, the target tissue to induce thefluorescence emission; initiating the capturing, by the camera, the timeseries of fluorescence input data from the fluorescence emission,wherein the camera is configured to receive fluorescence images forfluorescence medical imaging.
 2. The method of claim 1, wherein thefluorescence medical imaging comprises quantitative fluorescenceimaging, qualitative fluorescence imaging, or a combination thereof. 3.The method of claim 1, comprising, in response to detecting that thecurrent alignment is not aligned with the predefined alignment, ceasingthe illuminating and the capturing.
 4. The method of claim 1, whereininducing fluorescence emission comprises inducing fluorescence emissionfrom an endogenous fluorophore, from an exogenous fluorescence imagingagent, or a combination thereof.
 5. The method of claim 4, wherein theexogenous fluorescence imaging agent comprises indocyanine green (ICG).6. The method of claim 1, further comprising quantifying thefluorescence input data.
 7. The method of claim 1, further comprising,while the current alignment is aligned with the predefined alignment,acquiring the real-time video and processing the real-time video toquantify the target tissue.
 8. The method of claim 7, wherein theacquiring and processing of the real time video is initiated in responseto detecting that the current alignment is aligned with the predefinedalignment.
 9. The method of claim 7, wherein quantifying the targettissue comprises calculating a dimension of the target tissue.
 10. Themethod of claim 7, further comprising, in response to detecting that thecurrent alignment is not aligned with the predefined alignment, ceasingthe acquiring and processing of the real-time video.
 11. The method ofclaim 1, wherein receiving the reference image comprises receiving thereference image from a camera.
 12. The method of claim 1, whereinreceiving the reference image comprises retrieving stored datarepresenting the reference image.
 13. The method of claim 1, wherein thereference image is a white light image, a white light-derived image, afluorescence image, a fluorescence-derived image, or a combination ofany one or more thereof.
 14. The method of claim 1, wherein processingthe reference image to generate the alignment reference image comprisesapplying transparency to the reference image.
 15. The method of claim 1,further comprising displaying an alignment indicator on the display,wherein the alignment indicator indicates a difference between thecurrent alignment and the predefined alignment, and wherein thealignment indicator is distinct from the real-time video and thealignment reference image.
 16. The method of claim 15, whereinindicating a difference between the current alignment and the predefinedalignment comprises displaying a line between a first portion of thealignment reference image depicting a portion of the anatomical regionand a corresponding portion of the real-time video depicting the portionof the anatomical region.
 17. The method of claim 16, wherein the lineindicates a direction in which the current alignment should be adjustedto align with the predefined alignment.
 18. The method of claim 1,further comprising, in response to detecting that the current alignmentis aligned with the predefined alignment, displaying on the display anotification that the current alignment is aligned with the predefinedalignment, wherein the notification is distinct from the real-time videoand the alignment reference image.
 19. The method of claim 1, furthercomprising, in response to detecting that the current alignment isaligned with the predefined alignment, providing one of an auditory anda haptic notification that the current alignment is aligned with thepredefined alignment.
 20. The method of claim 1, wherein the referenceimage comprises one or both of a white light image and a processedimage.
 21. The method of claim 1, wherein the real-time video comprisesone or both of a white light image and a processed image.
 22. The methodof claim 1, further comprising applying an algorithm to determine thatthe current alignment is aligned with the predefined alignment.
 23. Themethod of claim 22, wherein applying the algorithm comprises calculatingan image transformation matrix including a translation and a rotationfor aligning the current alignment and the predefined alignment.
 24. Afluorescence imaging agent for use in the method of claim 1 for aligningthe camera.
 25. The fluorescence imaging agent of claim 24, whereinaligning the camera comprises aligning for blood flow imaging, tissueperfusion imaging, lymphatic imaging, or a combination thereof.
 26. Thefluorescence imaging agent of claim 25, wherein the blood flow imaging,tissue perfusion imaging, and/or lymphatic imaging comprises blood flowimaging, tissue perfusion imaging, and/or lymphatic imaging during aninvasive surgical procedure, a minimally invasive surgical procedure, orduring a non-invasive surgical procedure.
 27. The fluorescence imagingagent of claim 26, wherein the invasive surgical procedure comprises acardiac-related surgical procedure or a reconstructive surgicalprocedure.
 28. The fluorescence imaging agent of claim 27, wherein thecardiac-related surgical procedure comprises a cardiac coronary arterybypass graft (CABG) procedure.
 29. The fluorescence imaging agent ofclaim 28, wherein the CABG procedure is on pump or off pump.
 30. Themethod of claim 1, wherein the camera is mounted on a camera arm.
 31. Analignment system comprising: a display; a camera; a processor; andmemory storing instructions that, when executed by the processor, causethe processor to: receive a reference image of an anatomical region of asubject, the anatomical region comprising a target tissue; process thereference image to generate an alignment reference image; display thealignment reference image on a display concurrently with real-time videoof the anatomical region acquired from the camera; update the displayedreal-time video to reflect adjustments to a current alignment of thecamera relative to the anatomical region of the subject; and display thereal-time video and the alignment reference image as overlaid with oneanother when the current alignment of the camera is aligned with apredefined alignment associated with the reference image; determine thatthe current alignment is aligned with the predefined alignment, whereinthe determination is performed before initiating illuminating, by alight source, of the target tissue to induce fluorescence emission andis performed before initiating capturing, by the camera, of a timeseries of fluorescence input data from the fluorescence emission; inresponse to the determining that the current alignment is aligned withthe predefined alignment, the instructions further cause the processorto: initiate the illumination, by the light source, of the target tissueto induce the fluorescence emission; initiate the capture, by thecamera, of the time series of fluorescence input data from thefluorescence emission, wherein the camera is configured to receivefluorescence images for fluorescence medical imaging.
 32. The system ofclaim 31, wherein the fluorescence images are for fluorescence imaging,the fluorescence imaging comprising quantitative fluorescence imaging,qualitative fluorescence imaging, or a combination thereof.
 33. Thesystem of claim 31, wherein the instructions cause the processor to, inresponse to detecting that the current alignment is not aligned with thepredefined alignment, cease the illuminating and the capturing.
 34. Thesystem of claim 31, wherein inducing fluorescence emission comprisesinducing fluorescence emission from an endogenous fluorophore, from anexogenous fluorescence imaging agent, or a combination thereof.
 35. Thesystem of claim 34, wherein the exogenous fluorescence imaging agentcomprises indocyanine green (ICG).
 36. The system of claim 35, whereinthe instructions cause the processor to quantify the fluorescence inputdata.
 37. The system of claim 31, wherein the instructions cause theprocessor to, while the current alignment is aligned with the predefinedalignment, acquire the real-time video and process the real-time videoto quantify the target tissue.
 38. The system of claim 37, wherein theacquiring and processing of the real time video is initiated in responseto detecting that the current alignment is aligned with the predefinedalignment.
 39. The system of claim 37, wherein quantifying the targettissue comprises calculating a dimension of the target tissue.
 40. Thesystem of claim 37, wherein the instructions cause the processor to, inresponse to detecting that the current alignment is not aligned with thepredefined alignment, cease the acquiring and processing of thereal-time video.
 41. The system of claim 31, wherein receiving thereference image comprises receiving the reference image from a camera.42. The system of claim 31, wherein receiving the reference imagecomprises retrieving stored data representing the reference image. 43.The system of claim 31, wherein the reference image is a white lightimage, a white light-derived image, a fluorescence image, afluorescence-derived image, or a combination of any one or more thereof.44. The system of claim 31, wherein processing the reference image togenerate the alignment reference image comprises applying transparencyto the reference image.
 45. The system of claim 31, wherein theinstructions cause the processor to display an alignment indicator onthe display, wherein the alignment indicator indicates a differencebetween the current alignment and the predefined alignment, and whereinthe alignment indicator is distinct from the real-time video and thealignment reference image.
 46. The system of claim 45, whereinindicating a difference between the current alignment and the predefinedalignment comprises displaying a line between a first portion of thealignment reference image depicting a portion of the anatomical regionand a corresponding portion of the real-time video depicting the portionof the anatomical region.
 47. The system of claim 46, wherein the lineindicates a direction in which the current alignment should be adjustedto align with the predefined alignment.
 48. The system of claim 47,wherein the instructions cause the processor to, in response todetecting that the current alignment is aligned with the predefinedalignment, display on the display a notification that the currentalignment is aligned with the predefined alignment, wherein thenotification is distinct from the real-time video and the alignmentreference image.
 49. The system of claim 31, wherein the instructionscause the processor to, in response to detecting that the currentalignment is aligned with the predefined alignment, provide one of anauditory and a haptic notification that the current alignment is alignedwith the predefined alignment.
 50. The system of claim 31, wherein thereference image comprises one or both of a white light image and aprocessed image.
 51. The system of claim 31, wherein the real-time videocomprises one or both of a white light image and a processed image. 52.The system of claim 31, wherein the instructions cause the processor toapply an algorithm to determine that the current alignment is alignedwith the predefined alignment.
 53. The system of claim 52, whereinapplying the algorithm comprises calculating an image transformationmatrix including a translation and a rotation for aligning the currentalignment and the predefined alignment.
 54. A kit for aligning a camera,the kit comprising the system of claim 31 and a fluorescence imagingagent.
 55. A fluorescence imaging agent for use in the system of claim31 for aligning the camera.
 56. The system of claim 31, comprising acamera arm on which the camera is mounted.
 57. A non-transitory computerreadable storage medium storing instructions, wherein the instructionsare executable by a system having a display, a camera, and a processorto cause the system to: receive a reference image of an anatomicalregion of a subject, the anatomical region comprising a target tissue;process the reference image to generate an alignment reference image;display the alignment reference image on a display concurrently withreal-time video of the anatomical region acquired from the camera;update the displayed real-time video to reflect adjustments to a currentalignment of the camera relative to the anatomical region of thesubject; and display the real-time video and the alignment referenceimage as overlaid with one another when the current alignment of thecamera is aligned with a predefined alignment associated with thereference image; determine that the current alignment is aligned withthe predefined alignment, wherein the determination is performed beforeinitiating illuminating, by a light source, of the target tissue toinduce fluorescence emission and is performed before initiatingcapturing, by the camera, of a time series of fluorescence input datafrom the fluorescence emission; in response to the determining that thecurrent alignment is aligned with the predefined alignment, theinstructions further cause the processor to: initiate the illumination,by the light source, of the target tissue to induce the fluorescenceemission; initiate the capture, by the camera, of the time series offluorescence input data from the fluorescence emission, wherein thecamera is configured to receive fluorescence images for fluorescencemedical imaging.
 58. The non-transitory computer readable storage mediumof claim 57, wherein the fluorescence images are for fluorescenceimaging, the fluorescence imaging comprising quantitative fluorescenceimaging, qualitative fluorescence imaging, or a combination thereof. 59.The non-transitory computer readable storage medium of claim 57, whereinthe instructions cause the processor to, in response to detecting thatthe current alignment is not aligned with the predefined alignment,cease the illuminating and the capturing.
 60. The non-transitorycomputer readable storage medium of claim 57, wherein inducingfluorescence emission comprises inducing fluorescence emission from anendogenous fluorophore, from an exogenous fluorescence imaging agent, ora combination thereof.
 61. The non-transitory computer readable storagemedium of claim 60, wherein the exogenous fluorescence imaging agentcomprises indocyanine green (ICG).
 62. The non-transitory computerreadable storage medium of claim 61, wherein the instructions cause theprocessor to quantify the fluorescence input data.
 63. Thenon-transitory computer readable storage medium of claim 57, wherein theinstructions cause the processor to, while the current alignment isaligned with the predefined alignment, acquire the real-time video andprocess the real-time video to quantify the target tissue.
 64. Thenon-transitory computer readable storage medium of claim 63, wherein theacquiring and processing of the real time video is initiated in responseto detecting that the current alignment is aligned with the predefinedalignment.
 65. The non-transitory computer readable storage medium ofclaim 63, wherein quantifying the target tissue comprises calculating adimension of the target tissue.
 66. The non-transitory computer readablestorage medium of claim 63, wherein the instructions cause the processorto, in response to detecting that the current alignment is not alignedwith the predefined alignment, cease the acquiring and processing of thereal-time video.
 67. The non-transitory computer readable storage mediumof claim 57, wherein receiving the reference image comprises receivingthe reference image from a camera.
 68. The non-transitory computerreadable storage medium of claim 57, wherein receiving the referenceimage comprises retrieving stored data representing the reference image.69. The non-transitory computer readable storage medium of claim 57,wherein the reference image is a white light image, a whitelight-derived image, a fluorescence image, a fluorescence-derived image,or a combination of any one or more thereof.
 70. The non-transitorycomputer readable storage medium of claim 57, wherein processing thereference image to generate the alignment reference image comprisesapplying transparency to the reference image.
 71. The non-transitorycomputer readable storage medium of claim 57, wherein the instructionscause the processor to display an alignment indicator on the display,wherein the alignment indicator indicates a difference between thecurrent alignment and the predefined alignment, and wherein thealignment indicator is distinct from the real-time video and thealignment reference image.
 72. The non-transitory computer readablestorage medium of claim 71, wherein indicating a difference between thecurrent alignment and the predefined alignment comprises displaying aline between a first portion of the alignment reference image depictinga portion of the anatomical region and a corresponding portion of thereal-time video depicting the portion of the anatomical region.
 73. Thenon-transitory computer readable storage medium of claim 72, wherein theline indicates a direction in which the current alignment should beadjusted to align with the predefined alignment.
 74. The non-transitorycomputer readable storage medium of claim 73, wherein the instructionscause the processor to, in response to detecting that the currentalignment is aligned with the predefined alignment, display on thedisplay a notification that the current alignment is aligned with thepredefined alignment, wherein the notification is distinct from thereal-time video and the alignment reference image.
 75. Thenon-transitory computer readable storage medium of claim 57, wherein theinstructions cause the processor to, in response to detecting that thecurrent alignment is aligned with the predefined alignment, provide oneof an auditory and a haptic notification that the current alignment isaligned with the predefined alignment.
 76. The non-transitory computerreadable storage medium of claim 57, wherein the reference imagecomprises one or both of a white light image and a processed image. 77.The non-transitory computer readable storage medium of claim 57, whereinthe real-time video comprises one or both of a white light image and aprocessed image.
 78. The non-transitory computer readable storage mediumof claim 57, wherein the instructions cause the processor to apply analgorithm to determine that the current alignment is aligned with thepredefined alignment.
 79. The non-transitory computer readable storagemedium of claim 78, wherein applying the algorithm comprises calculatingan image transformation matrix including a translation and a rotationfor aligning the current alignment and the predefined alignment.
 80. Thenon-transitory computer readable storage medium of claim 57, wherein thecamera is mounted on a camera arm.